Pyridazine derivatives as novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors

Article abstract of DOI:10.1002/jhet.5570420306

Acyl-CoA:cholesterol acyltransferase (E.C.2.3.1.26, ACAT) is a microsomial enzyme that catalyses the formation of cholesteryl esters by acylation of cholesterol with long chain fatty acylCoA [1]. ACAT plays important roles in cellular homeostasis and in t

Full text of DOI:10.1002/jhet.5570420306

April 2005
395
Pyridazine derivatives as novel acyl-CoA:cholesterol
acyltransferase (ACAT) inhibitors
Arianna Gelain
Istituto di Chimica Farmaceutica e Tossicologica, Viale Abruzzi 42, 20131 Milano (Italy)
Acyl-CoA:cholesterol acyltransferase (E.C.2.3.1.26, ACAT) is a microsomial enzyme that catalyses the
formation of cholesteryl esters by acylation of cholesterol with long chain fatty acylCoA [1].
ACAT plays important roles in cellular homeostasis and in the early stages of atherosclerosis.
Therefore, ACAT inhibitors have been identified as useful agents in the treatment of hypercholes-
terolemia, atherosclerosis and coronary diseases [2]. In addition, recently their application has been pro-
posed for Alzheimer’s disease [3].
J. Heterocyclic Chem., 42, 395 (2005).
In mammalian species two isoforms are present: though
their role is not completely clarified, their distribution
seems to be peculiar: in particular, ACAT1 is predominant
in human liver, macrophage and adrenal gland while
ACAT2 is mainly present in the intestine [4].
related compounds, in which the imidazole ring was sub-
stituted by a pyridazine ring ( Fig.2).
The first studies on ACAT inhibitors were performed in
the early 70s. Since that time a large number of ACAT
inhibitors have been discovered and it is possible to clas-
sify these compounds in two major groups, on the basis of
their chemical structure:
Figure 2
•
•
Fatty acid anilide derivatives
Urea derivatives
Such compounds would retain several main characteris-
tics of ACAT inhibitors, namely a long alkyl chain linked
through an heteroatom to an heterocycle, in turn substi-
tuted by an ortho-biphenyl system.
Several representative examples are:
•
•
among fatty acids anilide derivatives: CI-976 [5], F12511
[6], TEI 6522 [7].
among urea derivatives: PD132301-2 [8], FR190809
[9], YM 17E [10].
We considered nitrogen, oxygen and sulphur in different
oxidation states as linkers between the heterocycle and
alkyl chain. Compounds where X = NH, NHCO and O
were synthesized following Scheme 1. The known piri-
dazin-3-one in turn obtained starting from desoxybenzoin
[14], was converted into the corresponding chloropyri-
dazine that under appropriate conditions gave the desired
products [15].
In addition, several interesting compounds not belong-
ing to the previous classes have been considered. Among
others CI-1011(Avasimibe) [11] and a series of 4,5-
diphenylimidazole derivatives, e.g. RP70676 [12],
RP73163 [13]. In particular, the latter attracted our atten-
tion (Fig.1).
Figure 1
In fact, given the interest of our group for the pyridazine
moiety, whose biological properties are well known, we
thought it challenging to study a class of structurally

396
Vol. 42
Scheme 1
a)
a)
POCl , ∆
3
for X=NH: RNH , ∆
2
for X=NHCO: 1) 30% aq. NH
2) RCOCl, r.t.
3
for X=O : RoNa, toluene, ∆
Scheme 2
a) R(CH ) Br, K CO , r.t.
2 n
2
3
b) CH COOH, 30% H O , r.t.
3
2 2
c) m.CPBA, r.t.
Table 1
When X = S, the pyridazin-3-one was first converted
into the corresponding thioderivative by treatment with
Lawesson’s reagent, then reacted with the suitable alkyl
bromide. Oxidation of the sulfides gave the corresponding
sulfoxides and solfones [15] (Scheme 2).
All the compounds were tested on ACAT extracted
from rat liver microsomes. The natural inhibitor GERI-
BP001 M, which in this assay shows IC = 42 µM,
50
Compound
X
n
IC (µM) [a]
50
was used as reference compound (Table1). The best
activity was obtained when X = NH and n = 8 as in
derivative 6. Shortening the side chain to n = 5 (com-
pound 5) still retained good activity. On the contrary,
the insertion of an amide instead of the amino group in
3, brought about a complete loss of activity. The pres-
ence of any other heteroatom than nitrogen always led
to less potent compounds.
1
2
3
4
5
6
7
O
S
SO
SO
NH
NH
5
5
5
5
5
8
4
72
62
75
60
24
2
18
>300
NHCO
[a] IC evaluated toward ACAT extracted from rat liver microsomes
(reference compound GERI-BP00 M IC = 42 µM).
50
50

April 2005
397
Table 2
On these bases, the subsequent SAR studies were limited
to the following substrate (Fig. 3). In particular, we consid-
ered several parameters:
• length and nature of the side chain, e.g. linear, branched, cyclic
• introduction of an additional heterocycle in the side chain
• presence of substituents on the ortho-biphenyl system
• importance of the ortho-biphenyl system.
Compound
R
R
IC (µM) [a]
1
50
8
9
H
H
H
H
H
CH
H
cyclopentyl
cyclohexyl
cycloeptyl
phenyl
46
3.6
25
4.5
36
31
18
10
11
12
13
Ref.
4-CF -phenyl
3
cyclohexyl
n-nonyl
3
[a] IC evaluated toward ACAT extracted from rat liver microsomes
Figure 3
50
nitro/aminophenyl derivatives was initially considered.
Their synthesis is reported in Scheme 3.
Initially we investigated the substitution of the linear
alkyl side chain with a cyclic mojety [16]. As shown
(Table 2) compounds 9 and 11, which present an alkyl and
an aromatic six membered ring, respectively, showed the
best activity. However it should be noted that both con-
tracting (compound 8) and enlarging (compound 10) the
ring size as well as introducing a substituent on the phenyl
ring (12) caused a dramatic loss of activity. The same neg-
ative result was obtained by transforming the secondary
amine of 9 into a tertiary amine as in 13.
After nitration, the pyridazin-3-ones were converted into
their corresponding chloropyridazines that were reacted
with the required amine. The complex mixture obtained
was purified by chromatography and the attribution of the
1
structures was done mainly on H-NMR bases [17].
The results of the biological tests (Table 3), reported
only for two representative products, clearly showed that
the presence of electronwithdrawing groups (14) caused a
significant loss of activity, while the presence of an NH
These data seem to suggest that the enzyme has good
tolerance for electronic variations, but more stringent
steric constrains.
To evaluate the influence of the presence of sub-
stituents on the ortho-biphenyl system, a series of
2
group as in 15, restored it. The values of the molecular
electrostatic potentials, which were calculated both for the
substituted and unsubstituted terms, suggested that the
introduction of substituents that made the pyridazine ring
Scheme 3
a)
b)
c)
HNO /H SO , r.t.
3
2
4
POCl
∆
3,
RNH , ∆
2

398
Vol. 42
more electron-rich would lead to an enhancement of the
activity.
Finally, the role of the ortho-biphenylic system on this
series was evaluated, by eliminating alternatively the
5-and 6-phenyl. As can be seen in Table 5, while the
5-monosubstituted phenyl (20) retains some activity with
respect to the biphenyl (6), the 6-phenyl derivative (21) is
inactive.
Table 3
Table 5
Compound
R
R
% inh. [a] V (NH) V (N1) V (N2)
1
2
(at 200 mg/ml)
14
15
Ref.
p.NO m.NO
53.5
77.6
74.4
46.6
77.6
74.4
-51.4
-60.1
-65.9
-52.6
-62.6
-64.7
2
2
2
p.NH m.NO
2
H
H
Compound
R
R
IC (µΜ) [a]
1
50
[a] % inh. evaluated toward ACAT extracted from rat liver microsomes
20
21
6
H
Ph
H
105
N.A.
18
Ph
Ph
Ph
[a] IC evaluated toward ACAT extracted from rat liver microsomes.
50
On these bases, we synthesized and tested a series of
chloro and methoxy analogues [18] (Table 4). The signifi-
cant activity showed by 16 with respect to its unsubstituted
analogue seems to confirm our hypothesis, which is further
validated by the values of the MEP of this new series. In
addition, it should be noted that the chlorine atom of 16 is
probably important in the interaction with the active site of
the enzyme and that the length of the side chain might
modulate this interaction, as is evident if we compare 16
and its longest analogue 17. On the contrary, the length of
the chain does not affect the activity of the methoxy substi-
tuted compounds.
More recently, we have extended our investigations to a
new series of pyridazine derivatives, which present an ure-
ido mojety in their structure. The rational is based on the
observation that, as already seen, many very potent ACAT
inhibitors, e.g. DuP 128 [19] present this functionality
(Figure 4).
Table 4
Figure 4
Compound
R
R
IC (µM) [a] V(NH) V(N1) V(N2)
50
1
16
(CH ) CH
p.Cl
p.Cl
1.0
80
11
36.9 -62.8 -62.7
36.2 -62.7 -62.6
35.4 -65.3 -64.3
35.0 -65.4 -64.2
35.8 -66.4 -64.8
2 5
3
In analogy with the DuP model, we initially considered
17
(CH ) CH
2 8
3
18
(CH ) CH p.OCH
2 5 3
3
compounds where X = S, SO, SO ; however, on the bases
2
19
(CH ) CH p.OCH
8.9
24
2 8
3
3
3
of our previous results, we also considered compounds
where X = NH. The synthesis [20] of the first series was
accomplished according to Scheme 4.
Ref.
(CH ) CH
H
2 5
[a] IC evaluated toward ACAT extracted from rat liver microsomes.
50

April 2005
399
Scheme 4
a) NH (CH ) CH , toluene, ∆
d) CBr , PBr , r.t.
4 3
2
2 6
3
b) LiAlH , THF
e) 5-phenyl or 5,6biphenyl-
2H-pyridazin-3-thione, K CO , ∆
f) Oxone, r.t.
4
c) 2,4-difluoro-phenylisocyanate, 0°C
K C , ∆
2
3
2
3
Table 6
The thioderivatives were synthesized starting from
δ-valerolacton, that was converted into its hydroxyamide
and reduced to the corresponding amine. The latter reacted
with 2,5-diF-phenylisocianate to give the hydroxy-ureido
derivative and after substitution with PBr , the intermedi-
3
ate was reacted with the suitable pyridazin-3-one. The sul-
fides obtained, by oxidation gave the corresponding sul-
foxides and sulfones . The compounds were purified by
chromatography.
The results of the biological assays (Table 6) showed
that both the mono- and the biphenyl sulfides (22 and 25)
were slightly more active in comparison with their corre-
sponding sulfoxides and sulfones. It should be noted that,
as seen in the previous series, also in this case the 6-phenyl
does not seem to be important for activity.
However, once more the best results were obtained with
the substitution of S with N. In particular the 5,6-biphenyl
derivative 30 and the 6-phenyl 29 were the most active,
while the 5-phenyl was almost devoid of activity.
In conclusion, our investigations studied the effects of
the substitution of the imidazole ring by a pyridazine ring
in a series of potent ACAT inhibitors. Our hope was to find
better features for these compounds, which are quite often
accompanied by severe side effects.
Compound
X
S
SO
SO
S
SO
SO
R
R
% inh. [a]
(at 50 µg/ml)
1
22
23
24
25
26
27
28
29
30
H
H
H
Ph
Ph
Ph
H
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
H
75
62
67
75
65
70
46
89
87
2
2
NH
NH
NH
Ph
[a] % inh. evaluated toward ACAT extracted from rat liver microsomes
(reference compound GERI-BP001 M % inh. =83 )
Acknowledgements.
In particular, we evaluated modifications on the het-
eroatom, the side chain and the o-biphenyl system and we
evidenced several characteristics, that could represent an
interesting enhancement of knowledge on the essential
requirements for good inhibitors.
I would like to thank Prof. Giorgio Cignarella and
Prof. Daniela Barlocco (University of Milan) for their
guidance and support. I would also like to acknowledge
all the other research group members for their contribute
and collaboration.

400
Vol. 42
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